Modified EDTA selectively recognized Cu2+ and its application in the disaggregation of β-amyloid-Cu (II)/Zn (II) aggregates

Article abstract of DOI:10.1016/j.jinorgbio.2019.110929

The accumulation of the β-amyloid (Aβ) aggregates induced by Cu²⁺/Zn²⁺ in conjunction with toxicity is closely related to Alzheimer's disease (AD). Herein, we intended to improve the efficiency and selectivity of traditional chelator

Full text of DOI:10.1016/j.jinorgbio.2019.110929

JournalofInorganicBiochemistry203(2020)110929
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Modified EDTA selectively recognized Cu2+ and its application in the
disaggregation of β-amyloid-Cu (II)/Zn (II) aggregates
Renke Chang1, Xiang Chen1, Huijuan Yu, Guizhen Tan, Hongli Wen, Jianxin Huang, Zhifeng Hao⁎
School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The accumulation of the β-amyloid (Aβ) aggregates induced by Cu2+/Zn2+ in conjunction with toxicity is
closely related to Alzheimer's disease (AD). Herein, we intended to improve the efficiency and selectivity of
traditional chelator ethylenediaminetetraacetic acid (EDTA) combined with a fluorescent group 4-aminosalicylic
acid (4-ASA)to acquire a novel potential chelator 4,4′-((2,2′-(ethane-1,2-diylbis((carboxymethyl)azanediyl))bis
(acetyl))bis(azanediyl))bis(2-hydroxybenzoic acid) (EDTA-ASA) capable of disaggregating Aβ-Cu(II)/ Zn(II)
aggregates. EDTA-ASA combines 4-ASA as fluorophore and multidentate amino nitrogen, hydroxyl and carboxyl
groups to chelate Cu2+ from Aβ-Cu (II) aggregates. The specific selectivity of EDTA-ASA towards Cu2+ in Tris-
HCl buffer solution was investigated by fluorescence measurements. It exhibits high recognition towards Cu2+
with no significant interference of other competitive metal ions, which overcomes the deficiencies of EDTA.
Importantly, the binding sites and binding mode for Cu2+ were clarified through DFT calculations. The thio-
flavin-T (ThT) fluorescence analyses and transmission electron microscopy (TEM) results have revealed EDTA-
ASA exhibited an enhanced disaggregation capability on Aβ-Cu (II)/Zn (II) aggregates in comparison to EDTA.
The Cu2+ chelating affinity was sufficient for EDTA-ASA to sequester Cu2+ from Aβ-Cu (II) aggregates.
Modified EDTA
Cu2+ recognition
β-Amyloid (Aβ)
Alzheimer's disease
Aβ-Cu (II)/Zn (II) aggregates
Disaggregation
1. Introduction
high millimolar concentrations range of Cu2+ and Zn2+ are found in SP
[8]. Up to now, the treatment of AD faces the greatest challenges in that
Alzheimer's disease (AD) is the most common form of dementia in
the world and a devastating neurodegenerative disorder, it is incurable
so far [1]. The predominant cause of AD is a heated discussion. Cur-
rently, though multiple factors mutually influence each other, the most
prevailing hypothesis is still the amyloid cascade [2,3]. The amyloid
cascade proposes amyloid-β (Aβ) aggregation as the leading cause of
the disease. The misfolding of the extracellular Aβ accumulated in se-
nile plaques (SP) has been recognized as the dominant hallmark of AD
[4]. Aβ is a typical 39–43 residue polypeptide encompassing a C-
terminal hydrophobic domain and an N-terminal hydrophilic sequence.
Its interaction with Cu2+, Zn2+ facilitates Aβ aggregation, Aβ mis-
folding and reactive oxygen species (ROS) production [5–7]. Especially,
there are few competent therapeutic approaches to modulate related
metal ions and disaggregate Aβ-Cu (II)/Zn (II) aggregates.
Metal chelators have been applied in AD therapy research due to
their metal ion chelating ability and have been proven to be a critical
approach [9–12]. In the past few decades, plenty of chelators for metal
ions, especially for Cu2+ selective recognition, have been reported such
as ethylenediaminetetraacetic acid (EDTA) [13], quinolines [14], clio-
quinol (CQ) [15a], 5,7-dichloro-2-((dimethylamino)methyl) 8-quino-
linol (PBT-2) [16], calixarenes [17], curcumins [18], and rhodamines
[19,20]. Potential regulation of Cu2+-induced Aβ aggregation has been
shown in vitro and in vivo by using these metal chelators. Several
chelators including CQ and PBT2 have been employed in murine AD
Abbreviations: AD, Alzheimer's disease; Aβ, Amyloid-β; SP, Senile plaques; ROS, Reactive oxygen species; EDTA, Ethylenediaminetetraacetic acid; CQ, Clioquinol;
PBT-2, 5,7-dichloro-2-((dimethylamino)methyl) 8-quinolinol; 4-ASA, 4-aminosalicylic acid; EDTA-ASA, 4,4′-((2,2′-(ethane-1,2-diylbis ((carboxymethyl)azanediyl))-
bis(acetyl))bis(azanediyl))bis(2-hydroxybenzoic acid); PET, Photoinduced electron transfer; NMR, Nuclear magnetic resonance; ESI-MS, Electrospray ionization mass
spectra; FT-IR, Fourier transform infrared; UV–vis, Ultraviolet-visible; TEM, Transmission electron microscope; EDTA-DA, 4-[2-(2,6-dioxomorpholin-4-yl) ethyl]
morpholine-2,6-dione; Py, pyridine; DFT, Density functional theory; B3LYP, Becke-3-Lee-Yang-Parr; MOs, Molecular orbitals; HOMO, Highest Occupied Molecular
Orbital; LOMO, Lowest Unoccupied Molecular Orbital; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; ThT, Thioflavin T; CHEQ, Chelation-enhanced fluorescence
quenching; ISC, Intersystem crossing; Ka, Binding association constant; B-H, equation Benesi-Hildebrand equation
⁎ Corresponding author.
E-mail address: haozf@gdut.edu.cn (Z. Hao).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.jinorgbio.2019.110929
Received 20 June 2019; Received in revised form 14 November 2019; Accepted 15 November 2019
Availableonline23November2019
0162-0134/©2019ElsevierInc.Allrightsreserved.

R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
models and AD patients. CQ was proved to be capable of dissolving Aβ
deposits in transgenic mice and it can slow the cognitive decline asso-
ciated with AD in some cases, and PBT2 could decrease the levels of Aβ
in AD patients. Yet, all these metal chelators have not yielded promising
results. The unpredictable side effects of these metal chelators, in-
cluding drug-resistance and subacute myelo-optic neuropathy, limit
their universal clinical applications. Besides, most of these chelators
exhibited limited application due to problems such as complicated
synthetic procedures, poor water solubility, and high interference by
co-existing metal ions [21–24]. Therefore, bearing the above con-
siderations in mind, the functional chelator designs are still novel, and
useful methods. It is of great significance and necessary to develop a
novel metal chelator which exhibits high selectivity towards Cu2+ in
aqueous solution via a simply synthetic method.
symmetrical structure, which is hoped to provide a special configura-
tion and binding cavity for Cu2+. In addition, the groups of –COOH and
–OH on 4-ASA could increase the solubility in aqueous solution. The
-CONH extends the distance between the two functional groups thereby
adjusting the effect of photoinduced electron transfer (PET) of sec-
ondary amines of 4-ASA to EDTA group. Furthermore, the –COOH and
the –OH group in EDTA-ASA may have electrostatic interactions and
hydrogen bonds with the carbonyl groups of N-terminal residues at Aβ.
Besides the chelating interaction with Cu2+, EDTA-ASA is expected to
have other synergistic effects to disaggregate the Cu (II)/Zn (II)-medi-
ated Aβ aggregates. Collectively, EDTA-ASA is anticipated to have a
wide potential application as potential chelator agents in the field of AD
therapy.
Interestingly, the well-known chelator, EDTA, has attracted con-
siderable attention in recent years [25–27]. Despite its limitations in
clinical studies, its EDTA backbone inspires us to functionalize it further
to improve its possible properties. We have started to construct a
modified EDTA derivative as a functional metal chelator. 4-aminosa-
licylic acid (4-ASA) has been widely utilized for the treatment of in-
flammatory diseases since the 1940s [28–31]. ASA conjugates of EDTA
were reported as promising anti-inflammatory prodrugs [32]. The
compound 4-ASA possesses a fluorescent moiety which is useful for
probing metal ions. Furthermore, 4-ASA conjugates of EDTA derivative
as a chelator for selective recognition of Cu2+ in AD therapy has not
been reported [33,34]. From the above, we have herein proposed to
conjugate 4-ASA to EDTA with the amide linkage (-CONH) as a bridging
unit to acquire the desired chelator, which will show water solubility
improvement, strong fluorescence, and be capable for specific re-
cognition of Cu2+ in aqueous solution.
2. Experimental section
2.1. Materials and methods
All chemical reagents (analytic grade or molecular biology grade)
purchased commercially were available and can be used as is unless
otherwise specified. The solutions of metal cations were performed
from their corresponding salts, such as LiCl, NaCl, KCl, MgCl2·6H2O,
CaCl2·4H2O, AlCl3·6H2O, BaCl2, ZnCl2, AgNO3, MnCl2·4H2O, Pb(NO3)2,
CrCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, HgCl2, and CuCl2·3H2O. For the
investigation of anions on the effect of recognition, the different water-
soluble chloride, sulphate, nitrate, bromide, perchlorate, and acetate
salts of copper (CuCl2, CuSO4, Cu(NO3)2, CuBr2, Cu(ClO4)2, and Cu
(OAc)2) were used. The water used in this work is ultrapure water
(18.2 MΩ cm) deionized by the Milli-Q system. Aβ40 is the most pre-
dominant forms of Aβ in vivo. Aβ40 peptide (free acid terminal) was
commercially purchased from Sangon Biotech (Shanghai, China) and
kept at −20 °C. The sequence of amino acid for the Aβ40 is DAEFRH-
DSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. The nuclear mag-
netic resonance (NMR) spectra were employed on Bruker AVANCE III
HD 400 in D2O. Electrospray ionization mass spectra (ESI-MS) were
determined on a Shimadzu, Japan LCMS-2020 Liquid Chromatograph-
Mass Spectrometer using methanol as a solvent. The Fourier transform
infrared (FT-IR) spectra were determined ranging from 4000 to
400 cm−1 on a THEMOR-FILSHER iS50R instrument using KBr pellets.
We strategically designed and synthesized a modified EDTA deri-
vative 4,4′-((2,2′-(ethane-1,2-diylbis ((carboxymethyl)azanediyl)) bis
(acetyl)) bis(azanediyl))bis(2-hydroxybenzoic acid) (EDTA-ASA)
(scheme. 1). It is based on EDTA as receptor and modified with 4-ASA
as fluorophore. EDTA-ASA comprising both 4-ASA and EDTA-backbone
group could provide excellent coordinating sites, such as electron-rich
N (amino‑nitrogen, -NH-) and O (hydroxyl and carboxyl groups, –OH,
–COOH) atoms. Both the framework of EDTA and side cores of 4-ASA
have the potential to chelate Cu2+. EDTA-ASA presents a highly
Scheme 1. Schematic illustration of the design strategy of EDTA-ASA and its disaggregation in Aβ-Cu (II)/ Zn (II) aggregates.
2

R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
Ultraviolet-visible (UV–vis) spectra were measured on PerkinElmer
Lambda 950 spectrophotometer. Fluorescence spectra were recorded on
a HORIBA Jobin Yvon FluoroMax-4 Fluorescence Spectrometer. Both
absorption and emission spectra were acquired at ambient temperature.
All tests were performed in parallel three times, and data processing
were expressed as the mean and standard deviation. Transmission
electron microscope (TEM) analysis was performed on the Shimadzu,
Japan HT7700.
dissolved in DMSO with agitation to acquire a final concentration of
1.0 mg·mL−1. The stock solution of Aβ40 was diluted with Tris-HCl
buffer solution (10 mM, pH 7.4) to desired concentrations for the fol-
lowing experimental.
2.6. Thioflavin T (ThT) fluorescence study
ThT fluorescence investigations were employed with a fluorescence
spectrometer. In the inhibition studies, freshly prepared Aβ40 (25 μM)
in the presence or absence of Cu (II) or Zn (II) (25 μM) was mixed with
or without EDTA and EDTA-ASA (25 μM) and incubated at 37 °C with
constant agitation for 24 h. In the disaggregation studies, metal-free
and metal treated Aβ40 (25 μM) aggregates were generated by in-
cubating mixtures of freshly prepared Aβ40 (25 μM) in the presence or
absence of Cu (II) or Zn(II) (25 μM) at 37 °C with agitation. After 24 h,
the samples were treated with EDTA and EDTA-ASA (25 μM) and in-
cubated for another 24 h. After incubation, all of the samples were
treated with ThT for the following ThT studies (λex = 404 nm,
λem = 487 nm).
2.2. Synthesis of 4,4′-((2,2′-(ethane-1,2-diylbis((carboxymethyl)
azanediyl)) bis(acetyl))bis(azanediyl))bis(2-hydroxybenzoic acid) (EDTA-
ASA)
The 4-[2-(2,6-dioxomorpholin-4-yl) ethyl] morpholine-2,6-dione
(EDTA-DA) (2.56 g (0.01 mol)), and 4-ASA (3.06 g (0.02 mol)) were
combined in 20 mL DMF under nitrogen atmosphere. 8 mL of pyridine
(Py) was sequentially added dropwise to the mixed solution. The solid
was completely dissolved for 15 min, and the reaction was stirred at
30 °C for 24 h. After the reaction was completed, the reaction mixture
was poured into CHCl3 to give a white precipitate. The obtained pro-
duct was collected by filtration under vacuum and washed with anhy-
drous ethanol. After vacuum drying at 60 °C for 5 h, a white powder
was obtained. This white powder was dissolved in an appropriate
amount of NaOH and then was followed by acidification by adding
0.1 M HCl dropwise. The pure white precipitate of EDTA-ASA was fi-
nally obtained by vacuum filtration in a yield of 92%.
2.7. Transmission electron microscopy (TEM)
Samples for TEM were prepared by the following steps. The glow-
discharged grid (Formar/Carbon 300-mesh, Electron Microscopy
Sciences) was treated with the corresponding Aβ40, and Aβ40-Cu (II)/Zn
(II) aggregates samples (25 μM, 5 μL) for 2 min at ambient temperature.
Excess samples were removed with filter paper and then washed twice
with ultrapure water. Each grid dried for 30 min at room temperature.
Images from each sample were captured by a Shimadzu, Japan HT7700
(40–120 kV, Image rotation: maximum range X1000-X40000 magnifi-
cation).
2.3. UV–vis spectra and fluorescence measurements of EDTA-ASA with
metal ions
The metal ions stock solution was prepared in ultrapure water using
the corresponding metal salts. This stock solution of EDTA-ASA was
prepared and diluted to desired concentration with Tris-HCl buffer so-
lution (10 mM, pH 7.4). Both UV–vis spectrum and fluorescence
emission spectrum were performed according to the following proce-
dures. In the UV–visible experiment, the EDTA-ASA solution was
placed in a quartz cell with an optical path of 1.0 cm to achieve an
absorption spectrum. For emission experiment, the solution of EDTA-
ASA 2000 μL was filled in a quartz cell with 1.0 cm optical path, the
solution of different metal ions was added and gradually mixed with a
micropipette for 3 min. The fluorescence titration experiment was
measured by mixing EDTA-ASA with a different molar ratio of Cu2+
solution.
3. Results and discussion
3.1. Synthesis and general characterization of EDTA-ASA
EDTA-ASA was synthesized in 92% yield via one-step reaction from
the starting materials EDTA-DA and 4-ASA respectively (as illustrated
in Scheme. 2). The structure of EDTA-ASA was well characterized by
ESI-MS, FT-IR, 1H NMR, and 13C NMR. All data match well with the
corresponding structure.
3.2. The spectroscopic studies of EDTA-ASA towards Cu2+ recognition
2.4. Computational details
In the UV–vis spectra, EDTA-ASA showed two main absorption
bands located at 260 nm and 302 nm in Tris-HCl buffer solution (Fig.
S5, ESI†). The UV–vis spectrum of 4-ASA exhibited a similar major
absorption band at 263 nm and 298 nm in Tris-HCl buffer solution (Fig.
S6, ESI†). The maximum absorbance band located at 260 nm corre-
sponds to benzene ring π-π* transition and the band centered at 302 nm
is attributed to the charge transition from benzene ring to an amino
group. Therefore, we choose the band centered at 260 nm as the ex-
citation wavelength in the subsequent fluorescent studies.
Density functional theory (DFT) theoretical calculation was em-
ployed to investigate the binding mode between EDTA-ASA and Cu2+
,
based on the 6-31G* in conjuration with Becke-3-Lee-Yang-Parr
(B3LYP) basis set [35–37]. The 6-31G* basis set was selected for non-
metallic elements such as the C, H, O, and N atoms; the SDD basis set
was for the Cu atom. The structures of EDTA-ASA and EDTA-ASA-Cu
complex were optimized with the DFT calculations. Vibrational fre-
quencies of the structure have been analyzed to confirm the minima
optimized structures. All theoretical calculations were accomplished on
the Gaussian 09 programs. The molecular orbitals (MOs) of EDTA-ASA
and EDTA-ASA-Cu were visualized and plotted with the Gauss-View
program.
To investigate the selectivity of EDTA-ASA, the tested metal ions
including Cu2+, Zn2+, Li+, Na+, K+, Mg2+, Ca2+, Al3+, Ba2+, Ag+
,
Mn2+, Pb2+, Cr3+, Co2+, Ni2+ and Hg2+ were employed in our study.
Fig. 1 shows the fluorescence emission spectra of EDTA-ASA in the
2.5. HFIP treatment of Aβ40
The initial powder of Aβ40 was dissolved in cooled 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) with constant agitation for 6 h to obtain a
homogeneous Aβ40 monomeric solution. The solution was blowing with
argon gas stream to evaporate the HFIP. The obtained thin white films
of the Aβ40 monomer were stored at −20 °C. Monomeric films were
Scheme 2. The synthesis procedure of EDTA-ASA
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R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
indicated the formation of 1:1 chelation ratio of EDTA-ASA and Cu2+
.
The 1:1 binding mode between EDTA-ASA and Cu2+ was also estab-
lished using Job's plot methods. The fluorescence emission intensity got
a minimum when the molar ratio fraction was 0.5, which further ver-
ified a 1:1 stoichiometry of EDTA-ASA and Cu2+. Cu2+ may be che-
lated at the N (-NH-, amino‑nitrogen) and O (-COOH, carboxyl group)
atoms of EDTA-ASA to satisfy the need for saturated 1:1 coordination
by forming stable EDTA-ASA-Cu complex. Theoretical calculations
based on DFT principles were employed to better understand the phe-
nomenon of fluorescence quenching and the chelation interaction be-
tween EDTA-ASA and Cu2+ in the following discussions.
The binding association constant (Ka) is used to evaluate the stabi-
lity of interaction between the chelator and metal ions together in a
solution. We adopted the Benesi-Hildebrand eq. (B–H equation) to de-
termine the Ka of EDTA-ASA and Cu2+ in Tris-HCl buffer solution [45].
There is a great linear relationship with the R2 value of 0.9984 in
Fig. 2(b). The Ka value was counted as 6.852 × 107 M−1 for EDTA-
ASA-Cu complex, indicating a robust binding association between
EDTA-ASA and Cu2+. Moreover, the EDTA-ASA-Zn complex showed
the same 1:1 stoichiometry, and the Ka was calculated to be
9.765 × 105 M−1, which is far below that of EDTA-ASA-Cu complex
(Fig. S9-S11, ESI†).
Fig. 1. Fluorescence spectrum of EDTA-ASA (10 μM) on addition of different
metal ions (10 μM) of Cu2+, Zn2+, Li+, Na+, K+, Mg2+, Ca2+, Al3+, Ba2+
,
Competition experiments study further assessed the specificity of
EDTA-ASA for Cu2+. as shown in Fig. 3(a), there was almost no sig-
nificant decrease in fluorescence intensity when EDTA-ASA was treated
Ag+, Mn2+, Pb2+, Cr3+, Co2+, Ni2+ and Hg2+ in Tris-HCl buffer solution
(10 mM, pH 7.4). Image: Selective fluorescent response of EDTA-ASA in the
presence of metal ions (under UV light).
with 1 equal of other metal ions (Zn2+, Li+, Na+, K+, Mg2+, Ca2+
,
Al3+, Ba2+, Ag+, Mn2+, Pb2+, Cr3+, Co2+, Ni2+, and Hg2+). How-
ever, upon addition of another 1 equal Cu2+ to the mixed solution of
EDTA-ASA with other metal ions, an apparent fluorescence quenching
was noticed. It was apparent that the quenching of fluorescence aroused
by the mixture of Cu2+ with other metal ions was almost the same as
that induced by Cu2+ only. As shown in Fig. 3(b), after EDTA-ASA was
treated with of 1 equiv. Cu2+, other excess 10 equiv. of competitive
metal ions were added, there is almost no interference in fluorescence
signal. Therefore, these consequences demonstrated that other common
competitive metal ions do not significantly influence the recognition of
EDTA-ASA for Cu2+, which confirms that EDTA-ASA behaves high
selectivity for Cu2+ in Tris-HCl buffer solution.
presence of 1.0 equal different metal ions. Collectively, EDTA-ASA
alone had relatively strong fluorescence centered at near 403 nm in
Tris-HCl buffer solution. Upon addition with 1.0 equal Cu2+, while, the
fluorescence at 403 nm was almost wholly quenched. The fluorescence
changed from “on” to “off.” In contrast, the other metal ions including
Zn2+ failed to show any dramatic fluorescence change in fluorescence
behavior of EDTA-ASA. The UV–vis spectra of EDTA-ASA on addition of
different metal ions in Tris-HCl buffer solution were also employed (Fig.
S7, ESI†). For comparison purpose, we also investigated the fluores-
cence properties of control compound 4-ASA in Tris-HCl buffer solu-
tion. A negligible response of 4-ASA was observed towards all the tested
metal ions (Fig. S8, ESI†). Therefore, the results confirmed that EDTA-
ASA exhibited good selectivity and displayed a dynamic “on-off” be-
havior towards Cu2+ in Tris-HCl buffer solution.
The fluorescence emission spectra of EDTA-ASA with different
counter anions (SO42−, Cl−, NO3−, Br−, ClO4−, and OAc−) was in-
vestigated to determine the influence of interference anions on the re-
cognition efficiency. This result illustrated that these anions had almost
no interference with the recognition of Cu2+ (Fig. S12, ESI†). It is the
chelation of Cu2+ and EDTA-ASA that leads to the efficient fluores-
cence quenching of EDTA-ASA, these anions exhibit no distinct effect
on the recognition selectivity.
Notably, the efficient quenching of fluorescence indicated that
EDTA-ASA showed a specific response to Cu2+. Before coordination
with Cu2+, EDTA-ASA have strong fluorescence. However, when
EDTA-ASA was coordinated with Cu2+, the fluorescence is quenched,
which is probably due to the formation of a non-fluorescent complex
EDTA-ASA-Cu. This “on-off” switching could be explained with chela-
tion-enhanced fluorescence quenching (CHEQ) effect and PET-en-
hanced process [38–40]. These metal ions differ too much in many
ways such as the orbital shape, electron density, size of the metal ion,
and they can establish different coordination interactions. The metal
ions that failed to cause a significant change in fluorescence intensity
may be due to the inappropriate coordination conformation and un-
suitable ion radius. This phenomenon is following the case of d9 Cu (II),
it is the highest in the Irving-Williams series [41]. Moreover, due to the
Jahn-Teller effect, the complex EDTA-ASA-Cu has higher stability [42].
Furthermore, Cu2+ is paramagnetic with an empty shell, the intrinsic
paramagnetic nature of Cu2+ from spin-orbit coupling could actively
quench the emission of a nearby fluorophore via non-radiative inter-
system crossing (ISC) transition process [43,44].
The reversibility of the fluorescence response process of EDTA-ASA
towards Cu2+ was also explored with EDTA. After addition of excess
EDTA to the EDTA-ASA and Cu2+ mixed solution, the fluorescence
returned to the original fluorescence of EDTA-ASA, which strongly
reveals that the Cu2+ recognition is a reversible chelation process (Fig.
S13, S14, ESI†). These results may be connected with the different Ka
between the chelators and Cu2+. Tough its binding affinity of Cu2+
with EDTA-ASA (EDTA-ASA-Cu, Ka = 6.852 × 107 M−1, namely lg
Ka = 7.84) is not as good as EDTA (EDTA-Cu, namely lg Ka = 18.7), it
exhibits a high selectivity towards Cu2+ with much higher efficiency
than EDTA.
It is well known that the stability of the metal chelator is of sig-
nificance for further practical application. Thus, the interrelationship
between the fluorescence of EDTA-ASA and pH variation was in-
vestigated to confirm the optimum pH range in application. When pH is
in the range of 2–4, the fluorescence intensity of the fluorescence
spectrum is nearly zero (Fig. S15, ESI†). This is probably due to the
weak solubility of EDTA-ASA in Tris-HCl buffer solution. The fluores-
cence intensity of EDTA-ASA increased moderately with the change of
pH from 4 to 7 and maintained relative stability in the pH range of
The fluorescence titration investigations were used to explore the
response properties of EDTA-ASA to Cu2+. As is presented in Fig. 2(a),
EDTA-ASA displayed strong fluorescence at 403 nm. Upon adding
various molar of Cu2+ (0–1 equal) into EDTA-ASA, the fluorescence
intensity gradually decreased, and the fluorescence intensity nearly
remains unchanged even 1.0 equal Cu2+ was added. The results
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R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
Fig. 2. (a) Fluorescence spectra of EDTA-ASA with addition of various concentrations of Cu2+ (0–1 equal, the total concentration of EDTA-ASA and Cu2+ was
10 μM) in Tris-HCl buffer solution (10 mM, pH 7.4). Inset: the drawing is Job's plot of the complexation between the EDTA-ASA and Cu2+. (b) Benesi-Hildebrand plot
of EDTA-ASA-Cu complex in Tris-HCl buffer solution (10 mM, pH 7.4).
7–12, which is ascribed to the protonation and deprotonation of EDTA-
ASA with multiple hydroxyl and amino groups.
Furthermore, the influence of the time on the fluorescence intensity
of EDTA-ASA over the range from 0 to 72 h in Tris-HCl buffer solution
was also evaluated. The results reveal that the fluorescence intensity
barely changes in a 10% range within a period of time (Fig. S16, ESI†).
Namely, the aqueous solution of EDTA-ASA and its fluorescence
properties are relatively stable, which means it may overcome the
physiological fluctuations and maintain expected performances.
Fig. 4. Optimized energy-minimized structure of EDTA-ASA and EDTA-ASA-Cu
3.3. Theoretical calculations
(C, N, O H, and Cu atom are represented as grey, blue, red, white-grey, and
orange respectively).
The outstanding performances of EDTA-ASA in the recognition of
Cu2+ drive us to explore its binding mode. Based on the results of
appropriate space to accommodate the Cu2+ and a cavity-like structure
fluorescence titration experiments and Job's plot, we hereby got the
was generated to tether Cu2+ successfully.
optimized energy-minimized structures of EDTA-ASA and EDTA-ASA-
In addition to the localization of the MOs (molecular orbital),
Cu by DFT optimization simulation calculations (as shown in Fig. 4).
EHOMO, ELUMO, and the HOMO (the highest occupied molecular orbital)-
Fig. S17, and Fig. S18 ESI† illustrated the energy optimization curves
LUMO (the lowest unoccupied molecular orbital) band gap
(∆E = ELOMO-EHOMO) are commonly used parameters to interpret the
electronic properties and chelation behavior. Therefore, the orbital
energies and the spatial electronic cloud distributions of the HOMO and
the LUMO of EDTA-ASA and EDTA-ASA-Cu complexes were shown in
Fig. 5. The HOMO of EDTA-ASA is primarily delocalized on the fluor-
ophore 4-ASA part, the LUMO is the same as the distribution of HOMO.
There is no electron transfer, And the PET process is prohibited, EDTA-
ASA showed fluorescence. However, when EDTA-ASA is converted to
EDTA-ASA-Cu after coordinating with Cu2+, the HOMO of EDTA-ASA-
Cu is mostly delocalized on the Cu2+ and nearby EDTA part, while the
LUMO is distributed on the fluorophore 4-ASA part. Under this
under the best binding mode between EDTA-ASA and Cu2+. The op-
timized structure of EDTA-ASA showed that it had a symmetrical plane
containing the fluorophore and the receptor. The Cu2+ coordination
changes the conformational states of EDTA-ASA. The structure of
EDTA-ASA-Cu displays that the Cu2+ binds to EDTA-ASA at the amino
nitrogen atoms and the carboxyl groups of core framework EDTA
through four coordination sites. And, the Cu − O bond lengths are
2.03991 Å and 2.13863 Å, the Cu − N bond lengths are 2.03111 Å and
2.03989 Å, respectively, which means a strong coordination bond in-
teraction between EDTA-ASA and Cu2+. The coordination distances are
suitable for Cu2+ binding and utilized exclusively for Cu2+. These data
indicated that the structure of EDTA-ASA could effectively provide
Fig. 3. (a) The fluorescence intensity at 403 nm of EDTA-ASA (10 μM) with metal ions in Tris-HCl buffer solution (10 mM, pH 7.4) and then with addition of equal
molar of Cu2+. (b) the fluorescence intensity at 403 nm of EDTA-ASA (10 μM) with the addition of equal molar of Cu2+ and then addition of excess 10 equiv. of other
metal ions in Tris-HCl buffer solution (10 mM, pH 7.4).
5

R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
Fig. 6. The disaggregation of EDTA-ASA (Pink) and EDTA (Cyan) on corre-
sponding Aβ40 and Aβ40-Cu (II)/Zn (II) aggregates in Tris-HCl buffer solution
(10 mM, pH 7.4, λex 440 nm, λem 487 nm,) were monitored by ThT fluores-
cence assay.
Fig. 5. Optimized frontier molecular orbital profiles for EDTA-ASA and its
complex EDTA-ASA-Cu based on DFT (B3LYP/6-31G* set) calculation.
Zn2+ and Aβ40. It is generally assumed that the Cu2+ can coordinate
with Aβ40 at N-terminal residues, such as His6, His13, His14, and
thereby promote the aggregation of Aβ40. Likewise, Aβ40 could form a
1: 1 complex with Zn2+ [4,5,51]. When EDTA-ASA was added to the
sample of Aβ40-Cu (II)/Zn (II) aggregates, a decline in fluorescence was
circumstance, this corresponds to the electron cloud distribution of the
PET-enhanced process between the receptor and the fluorophore 4-
ASA.
The ∆E between HOMO and LUMO of EDTA-ASA and EDTA-ASA-
Cu was calculated as 0.22313 eV and 3.32115 eV, respectively. The ∆E
between HOMO and LUMO of the EDTA-ASA-Cu complex had in-
creased 3.09802 eV as compared to that of EDTA-ASA. Moreover, the
geometry energy of EDTA-ASA-Cu was lower than EDTA-ASA (Fig. S17,
Fig. S18, Table 1, ESI†). Therefore, the conversion of EDTA-ASA to
EDTA-ASA-Cu becomes more accessible to form a stable EDTA-ASA-Cu
complex by broadening the energy gap between HOMO and LUMO,
which is also in accordance with Fang, Kim, Anand, and Hou's re-
searches [46–49].
observed, which indicated that EDTA-ASA could inhibit the Cu2+
/
Zn2+-induced Aβ40 aggregation. Moreover, EDTA-ASA also dis-
aggregate Aβ40-Cu (II)/Zn (II) aggregates. It is worth noted that the
ability of EDTA-ASA to depolymerize Aβ40-Cu (II)/Zn (II) aggregates is
much stronger than the ability to inhibit aggregation, so EDTA-ASA was
evaluated for its disaggregation capacity on self-induced Aβ40, and
Cu2+/Zn2+-induced Aβ40 aggregates in subsequent investigations.
As shown in Fig. 6, the depolymerization rate of EDTA-ASA for
Aβ40-Cu (II) aggregates was 62.20%, which is 1.37-fold more effective
than EDTA. EDTA, a natural product that is known to disaggregate Aβ-
M (II) aggregates, was used as the reference compound [52]. We found
that EDTA disaggregate Aβ40-Cu (II) aggregates by a percentage similar
to previous reports. (26.22% vs 24.50% disaggregation). EDTA-ASA
was more effective at dissolving Aβ40-Cu (II) aggregates compared with
the control EDTA. Moreover, the consequence of EDTA-ASA to depo-
lymerize in Aβ40-Zn (II) aggregates is not as good as that of Aβ40-Cu (II)
aggregates. The ThT disaggregation results of EDTA-ASA and EDTA on
the corresponding Aβ40 aggregates are listed clearly in Table. 1.
TEM was exerted to directly study the morphology of corresponding
Aβ40 aggregates and visualize the disassembly effect of EDTA-ASA. As
shown in Fig. 7, regular linear fibers morphology and little-fibrillary
3.4. Potential application of EDTA-ASA in disaggregating Aβ40-Cu (II)/Zn
(II) aggregates
The ThT fluorescence study was explored to evaluate the ability of
EDTA-ASA to inhibit and disaggregate Cu2+/Zn2+-induced Aβ40 ag-
gregates (Aβ40-Cu (II)/Zn (II) aggregates). ThT is a benzothiazole
fluorescent dye with a high affinity for β-sheet amyloid fibers, and it
can be utilized to quantify the β-sheet content of Aβ aggregates [50]. As
is clearly shown in Fig. S19-S21 ESI†, there's no fluorescence signal of
the ThT in the fresh Aβ40 solution. The solution of self-aggregated Aβ40
sample only developed a limited increase in fluorescence intensity. In
contrast, in the presence of Cu2+/Zn2+, there is a significant increase of
approximately more than 4-fold in fluorescence signal, compared with
the self-aggregated Aβ40 aggregates, which suggest that Cu2+/Zn2+
could induce the aggregation of Aβ40 and the generation of the β-sheet
structure. Although precise interaction mechanism of the Cu2+/Zn2+
with Aβ40 is still unclear, it is generally conjectured that these may
aggregates were detected in the samples of the self-aggregated Aβ40
,
Cu2+-induced Aβ40 formed a denser aggregate than that of self-ag-
gregated Aβ40, larger aggregates were observed in Zn2+-induced Aβ40
aggregates. In the presence of EDTA-ASA, the large Aβ40-Cu (II) ag-
gregates were broken down into much smaller amorphous aggregates
compared with the EDTA. The morphology of Aβ40 eCu (II) aggregates
became smaller, and the structure was looser. All these findings are
consistent with the above ThT fluorescent analysis. Consequently, it is
assumed that the collapse of the Aβ40-Cu (II) aggregates is mainly due
to the capability of EDTA-ASA to chelate Cu2+, and thus loosening the
interactions of Cu2+ with the Aβ40. At pH 7.4, the logarithm of the Ka of
Aβ40 for Cu2+ (lgKa [Cu2+-Aβ] = 5.80) is 5.80 [53]. This value is much
lower compared with the affinity of EDTA-ASA for Cu2+ that has been
obtained in the above discussions, (lgKa [Cu2+-EDTA-ASA] =7.84).
Indeed, the affinity of EDTA-ASA to Cu2+is greater than that of Aβ40
(lgKa [Cu2+-Aβ] = 5.80, lgKa [Cu2+-EDTA-ASA] =7.84). Conse-
quently, EDTA-ASA can extract Cu2+ complexed by Aβ40 aggregates,
resulting in the disassembly of the Aβ40-Cu (II) aggregates. Also, the
result from the electrostatic and chelating interactions between Cu2+
/
Table 1
The disaggregation results of EDTA-ASA and EDTA on corresponding Aβ40 and
Aβ40-Cu (II)/Zn (II) aggregates.
Samples
DRa (%)
EDTA
EDTA-ASA
Aβ40 aggregates
19.60
26.20
8.20
0.23
0.21
44.20
62.20
32.30
0.31
Aβ40-Cu (II) aggregates
Aβ40 − Zn (II) aggregates
0.36
0.33
0.27
DRa stands for disaggregation rate.
6

R. Chang, et al.
JournalofInorganicBiochemistry203(2020)110929
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